MicroRNA-561-3p indirectly regulates the PD-L1 expression by targeting ZEB1, HIF1A, and MYC genes in breast cancer

Globally, breast cancer is the second most common cause of cancer-related deaths among women. In breast cancer, microRNAs (miRNAs) are essential for both the initiation and development of tumors. It has been suggested that the tumor suppressor microRNA-561-3p (miR-561-3p) is crucial in arresting the growth of cancer cells. Further research is necessary to fully understand the role and molecular mechanism of miR-561 in human BC. The aim of this study was to investigate the inhibitory effect of miR-561-3p on ZEB1, HIF1A, and MYC expression as oncogenes that have the most impact on PD-L1 overexpression and cellular processes such as proliferation, apoptosis, and cell cycle in breast cancer (BC) cell lines. The expression of ZEB1, HIF1A, and MYC genes and miR-561-3p were measured in BC clinical samples and cell lines via qRT-PCR. The luciferase assay, MTT, Annexin-PI staining, and cell cycle experiments were used to assess the effect of miR-561-3p on candidate gene expression, proliferation, apoptosis, and cell cycle progression. Flow cytometry was used to investigate the effects of miR-561 on PD-L1 suppression in the BC cell line. The luciferase assay showed that miRNA-561-3p targets the 3′-UTRs of ZEB1, HIF1A and MYC genes significantly. In BC tissues, the qRT-PCR results demonstrated that miR-561-3p expression was downregulated and the expression of ZEB1, HIF1A and MYC genes was up-regulated. It was shown that overexpression of miR-561-3p decreased PD-L1 expression and BC cell proliferation, and induced apoptosis and cell cycle arrest through downregulation of candidate oncogenes. Furthermore, inhibition of candidate genes by miR-561-3p reduced PD-L1 at both mRNA and protein levels. Our research investigated the impact of miR-561-3p on the expression of ZEB1, HIF1A and MYC in breast cancer cells for the first time. Our findings may help clarify the role of miR-561-3p in PD-L1 regulation and point to this miR as a potential biomarker and novel therapeutic target for cancer immunotherapy.


MiR-561-3p directly downregulate the expressions of MYC, HIF1A, and ZEB1 genes in BC cells
To confirm the hypothesis that overexpression of miR-561-3p could downregulate the expression of ZEB1, MYC, and HIF1A genes in BC cell lines, the miR-561-3p or scrambled oligonucleotide was transfected into MDA-MB-231, BT-549, and MCF-7 cells, and the level of MYC, HIF1A, and ZEB1 mRNA was measured by quantitative real-time PCR (Fig. 3, P < 0.01).The results showed that the overexpression of miR-561-3p is followed by a reduction in the level of ZEB1, MYC, and HIF1A mRNA in MDA-MB-231, BT-549, and MCF-7 cell line (Fig. 3A-C) (mean ± SE) compared to non-transfected cells.

MiR-561-3p arrested the cell cycle
A flow cytometry assay was used to evaluate the cell cycle arrest induced by miR-561-3p in MDA-MB-231, BT-549, and MCF-7 cell lines after 48 h of transfection.The results demonstrated that overexpression of miR-561-3p increased the G0/G1 phase fraction (P < 0.05) and decreased the S and G2/M phase fractions ) P < 0.05) (Fig. 6A, B).

Discussion
In BC, the bulk of malignant tumors are associated with the number of tumor-infiltrating lymphocytes (TILs) or immune gene expression signatures 45,46 .The malignant cells and somatic mutations that cause immunogenic death are associated with inferior outcomes and chemotherapy resistance; thus, an effective immune system is critical in producing neoantigens that elicit an adaptive immune response and will clear or keep the escaping tumor cells dormant 47 .Therefore, identifying molecular pathways involved in the development and progression of BC is crucial for the investigation of novel and practical diagnosis markers, as well as potential therapeutic targets, which are urgently needed to understand 48 .In the last few years, a new BC treatment based on ICI (immune checkpoint inhibitors) such as anti-PD-1/PD-L1 agents is a promising strategy.Better response to ICI (immune checkpoint inhibitor ) treatment may be predicted by PD-L1-positive status, non-liver metastases, firstline immunotherapy, and high TIL and CD8 + T-cell infiltration levels.Patients with PD-L1-positive tumor could gain more survival benefits from immune checkpoint therapy 49 .However, single-cell transcriptomic sequencing of breast cancer cells that metastasized to the liver and brain showed that the expression of the immunoreceptor inhibitory checkpoint genes, LAG3 and TIGIT, in T cells was higher than that of PDCD1 (PD-1).Based on these observations, individuals with liver and brain metastasis may benefit from therapeutic targeting of these two second-generation immune checkpoint receptor targets and their ligands (LGALS3 and NECTIN2) 50 .
Sabatier et al. analyzed 5454 BCs DNA microarrays and PD-L1 mRNA of 45 BC cell lines, and their finding showed PD-L1 overexpression in 20% of clinical samples and 38% of basal tumors, and they suggested that PD-L1 overexpression was related to decreased T cell cytotoxic immune response 51 .Muenst et al. assayed PD-L1 expression in 650 evaluable formalin-fixed BC sample cases, and they reported that 152 (23.4%) of the 650 BC specimens were PD-L1 positive 52 .
In March 2019, antibodies targeting PD-1/PD-L1, such as atezolizumab plus nab-paclitaxel, were approved by the Food and Drug Administration (FDA) to increase the survival rate in advanced TNBC patients with PD-L1-positive tumors.Soon, anti-PD-1/PD-L1 agents are going to make impressive achievements in BC therapy.However, combination therapeutic strategies, including immune checkpoint blockade and multi-agent chemotherapy to increase patients' survival rates, are often administered in the early and advanced stages 53,54 .
Recently, studies have suggested that disregulation of miRs plays vital roles in the initiation and progression of BC via targeting PD-L1 expression [2][3][4] .For example, Dastmalchi et al. reported that miR-424-5p inhibited proliferation and invasion of BC cells through targeting PD-L1 and modulating the PTEN/PI3K/AKT/mTOR signaling pathway; therefore, miR-424-5p is considered a tumor-suppressor miR in BC 2 .LanlanGao et al. showed that miR-873 could reduce the stemness and chemoresistance of BC cells by targeting PD-L1 expression, which eventually suppressed the PI3K/Akt and ERK1/2 pathways 4 .Azarbarzin et al. demonstrated that inhibition of cell proliferation and migration and apoptosis induction could happen in BC cells via miR-383-5p transfection.MiR-383-5p has a role as a tumor suppressor through an inhibitory effect on the PI3K/AKT/mTOR pathway and inhibiting tumor PD-L1 expression 55 .According to the study of Rasoolnezhad et al., miR-138-5p could induce apoptosis and cell cycle arrest, and inhibit migration and proliferation via targeting PD-L1 and then, PI3K/ AKT pathway in BC 56 .In another study, it was showed that the 3-UTR of both PD-L1 and LDHA genes have binding sites for miR-34a.These acted as ceRNAs (competitive endogenous RNAs) to promote the expression   and function of each other through regulation of miR-34a in TNBC 57 .In this study, we investigated the effect of miR-561-3p on proliferation, apoptosis, and cell cycle arrest via indirect targeting of PD-L1 in BC.Some studies have reported that miR-561-3p plays a crucial role in cancer progression.For example, Kun Qian et al. have studied the potential role of miR-561-3p in gastric cancer development, and their finding confirmed that miR-561-3p inhibits cell proliferation and invasion by downregulating c-MYC expression 40 .Karimi et al. demonstrated the inhibitory effect of miR-561-3p on glioblastoma cell proliferation and apoptosis via targeting c-MYC expression.This finding suggests that miR-561-3p is a promising agent for GBM (glioblastoma multiforme) treatment 43 .Liao et al. demonstrated that miR-561-3p has an inhibitory role on cell cycle and proliferation by targeting P-REX2a and then, regulating the PTEN/AKT signaling pathway in NSCLC 41 .
Currently, the effect of miR-561 has not yet been investigated in breast cancer.Here, for the first time, we investigated the functionality of this miR in the inhibition of breast cancer.In an unpublished study, Hajibabaei et al. found that MALAT1 knockdown by siRNA could prevent BC cell progression through the miR-561-3p/ TOP2A axis 58 .PD-L1 is regulated at the genomic, epigenetic, transcriptional, and post-translational levels 59 .In another study conducted by Hajibabaei et al. the direct inhibition of PD-L1 by miR-145 and miR-335 was discussed in breast cancer 44 and, in this study, miR-561-3p was used to indirectly target the PD-L1 gene via ZEB1, MYC, and HIF1A genes.First, we selected PD-L1-positive BC tissues and examined the expression of miR-561-3p in these specimens.We found that miR-561-3p was downregulated in tumor samples compared to normal, As we mentioned above, over expression of MALAT1 can be the probable reason for the downregulation of miR-561-3p in BC 58 .Using bioinformatics analysis, we demonstrated that ZEB1, MYC, and HIF1A have potential as novel targets for miR-561-3p.The dual-luciferase reporter assay confirmed that miR-561-3p could directly target the ZEB1, MYC, and HIF1A 3′-UTRs and inhibit their transcription.In the current study, the qRT-PCR results showed an opposite relationship between ZEB1, HIF1A, and MYC mRNA expression and miR-561-3p overexpression in BC tissues.Based on these results, miR-561-3p may play an important role as a tumor suppressor in BC.
In various types of human cancer, including colorectal 60 , cervical 61 , gastric 62 , and bladder cancer 63 , ZEB1 has been upregulated, and its overexpression has some effects on cellular processes, including cell growth, apoptosis, migration, invasion, metastasis, tumor development, and tumor progression 64,65 .In TNBCs (triple-negative BCs) www.nature.com/scientificreports/and basal-like BCs, the level of ZEB1 is higher than the luminal subtype 66 .Moreover, ZEB1 overexpression has a role in resistance in therapy, poor survival, and increased metastatic risk in BC 66 .The ZEB1 promoter is activated by the TGF-β signaling pathway (21 ).Therefore, the transition of cell phenotypes from CD44-low to CD44-high is increased, and then the growing generation of CSC signals occurs in BC 21 .Ataxia-telangiectasia mutated (ATM) kinase phosphorylates ZEB1 and leads to growing ZEB1 stabilization in response to DDR (DNA damage repair) 67 .In BC patients with ZEB1 overexpression, chemoresistance via inadequate responses to EPI (epirubicin) was observed 68 .The double-negative feedback loop between ZEB1 and the miR-200 axis plays a critical role in regulation of PD-L1 overexpression.The miR-200c expression can be inhibited in cancer cells by aberrant expression of EMT-factors, such as ZEB1 69 , and consequently, the expression of PD-L1 (as a target of miR-200 increased ) 20 .By creating EMT phenotypes and stem cell-like features in tumor cells, ZEB1 can play an important role in the development of BC.Therefore, inhibition of ZEB1 expression is a worthy suggestion for the treatment of BC 68,70 .
ZEB1 was a target of miR-601 and miR-448, and overexpression of this gene could reverse the suppressive effects of miR-601 and miR-448 on the development of BC 71,72 .It was showed that miR-409-3p targets ZEB1 to control the invasion and metastatic process of BC.This suggests that miR-409-3p could be a novel prognostic marker and therapeutic target for the treatment of BC metastasis 73 .
HIF1A can bind to HRE regions of the PD-L1 promoter and promotes the expression of PD-L1 26 .Meanwhile, inhibiting HIF1A signaling could reduce PD-L1 expression in multiple types of cancers 26,74 HIF1A activation promotes glycolytic metabolism, angiogenesis, and carcinogenesis 75 .Furthermore, HIF1A can overexpress LDH-A (lactate dehydrogenase) which thus facilitates the accumulation of lactate in the tumor microenvironment and promotes tumor invasiveness, drug resistance, and immune escape of tumor cells 76 .Excessive activity of hypoxiarelated pathways is correlated with the proliferation ability of CTCs (circulating tumor cells) in the brain and also blood-borne metastasis to the brain in women with advanced metastatic BC 77 , Therefore, HIF1A targeting could be a promising therapeutic approach for cancer.
Under hypoxic conditions, HIF-1A was upregulated in breast cancer cells, and overexpression of some miR-NAs such as miR-497 and miR-7641 can suppress this gene 78,79 .In a nude mouse xenograft tumor model, miR-497 suppressed tumor growth and decreased angiogenesis 78 .Down-regulating HIF-1A expression in breast cancer cells may be one of the mechanisms by which miR-7641 suppresses cancer stemness 79 .
MYC is a proto-oncogene in many cancers, including Burkitt's lymphomas, lung carcinoma, breast carcinoma, and colon carcinoma.Moreover, MYC, as a proto-oncogene, can regulate cell differentiation, proliferation, and apoptosis 80 .Amplification of MYC is present in 30-50% of high-grade BCs and is related to resistance in anticancer therapies; therefore, it is a useful predictive marker for drug and RFS (relapse-free survival ( 81 .MYC binds directly to the PD-L1 promoter region and, as a transcription factor, increases PD-L1 expression.MYC participates in PD-L1 regulation,interacts with BC stem cell markers such as CD44, CD24, and ALDH1, and plays a significant role in the regulation of the initiation and metastasis of BC 80 .MYC overexpression inhibits BRCA1's tumor suppressor and causes the development of basal-like BC; therefore, targeting MYC-regulated pathways provides a promising therapeutic strategy for BC 82 . The other study, examined the c-myc/miR-29b-3p/CDK6 axis, is thought to be responsible for downregulating miR-29b-3p by c-myc to enhance CDK6 activation and induce palbociclib resistance in breast cancer.This was done using both xenograft models and patient-derived tumor xenograft (PDTX) models.They proposed c-myc as a potential biomarker for breast cancer patients' susceptibility to palbociclib.Kinase inhibitors, such as palbociclib, aid in preventing or postponing the spread of cancer cells 83 .
In this study, the ability of miR-561-3p to decrease proliferation and induce apoptosis and cell cycle arrest was examined by transfecting miR-561-3p into MDA-MB-231, BT-549, and MCF-7 cell lines.The results demonstrated that miR-561-3p could downregulat the expression of ZEB1, HIF1A, and MYC genes.In this study, we examined the effects of miR-561-3p overexpression in BC in two ways.First, miR-561-3p could decrease the expression of PD-L1 by targeting the ZEB1, HIF1A, and MYC genes.Second, downregulation of PD-L1 by targeting some signaling pathways such as MAPK and PI3K/Akt.Also, targeting some transcriptional factors that are well known to reduce cancer development through boosting the immune response against cancer cells, inhibiting cell proliferation, inducing apoptosis 84 , and arresting the cell cycle 85 .On the other hand, downregulation of ZEB1, HIF1A, and MYC genes could prevent the development of BC by inhibiting cell proliferation and increasing apoptosis 20,64,65,75,80 .
The results of this study demonstrated that miR-561-3p may indirectly affect PD-L1 expression by degrading ZEB1, HIF1A, and MYC mRNA, stopping BC cell proliferation, and inducing apoptosis.Based on our results, targeting ZEB1, HIF1A, and MYC may be a novel strategy for BC therapy, and miR-561-3p is a novel and promising candidate for microRNA restoration therapy in BC patients.To attain more accurate outcomes for the analysis of the manifestation of the PD-L1 gene and its impact on the progression of BC, it is necessary to have more sample volumes.Moreover, it could be better to perform some supplementary tests, such as RNA sequencing for deep knowledge of the role of PD-L1, ZEB1, MYC, HIF1A gene expression in BC.

RNA extraction, reverse transcription, and RT-qPCR
Total RNA of BC cell lines, normal tissues, and tumor samples was extracted with QIAzol ® (Qiagen, Germany).cDNAs were synthesized by M-MuLV reverse transcriptase enzyme included in the cDNA synthesis kit (Fermentas, EP0351) according to the manufacturer's protocol.GAPDH was used as a housekeeping and normalizer for all target mRNAs.Each qPCR assay was performed in triplicate.RT-qPCR reactions were prepared in a final volume of 25μl containing 140ng of specific primers (Table 2) and 12.5μL SYBR green master mix (Life Technology, 4309155).RT-qPCR reaction was performed by the following program: enzyme activation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95°C for 20 s, annealing at 59 °C for 30 s, and extension at 72 °C for

Designing the stem-loop
The miR-561-3p sequence was obtained from the miRbase website (http:// www.miRba se.org/).To design the stem-loop structure with increased flexibility and necessary sensitivity, the sequence of Chen et al. was modified.Also, the loop was enlarged to design a universal reverse primer and TaqMan probe inside it and nucleotide replacement was performed to decrease the melting temperature of the stem part.All of these modifications added 14 nucleotides to the original sequence of miR-561-3p (Table2).In order to detect miR-561-3p in the BC normal and tumor tissues by real-time PCR, the complete sequence of miR-561-3p as the forward primer was used, and also U6 as the reference gene.All oligonucleotides were purchased from the Cinaclon Company (Iran, Tehran).

Luciferase reporter assay
In the first step, the 3′-UTRs of ZEB1, HIF1A, and MYC genes were amplified using specific primers (Table 3).The highlighted sequences in

MicroRNA transfection
miR-561-3p mimics and scrambled microRNA were obtained from GenePharma Co. (Shanghai, China).MDA-MB-231, BT-549 and MCF-7 cell lines were used as cells expressing PD-L1, and MCF-10 cell line was used as a negative control.The BC and negative control cell lines were transfected using HiPerFect Transfection Reagent (QIAGEN, Germany) according to the manufacturer's protocol.Transfected cell lines were harvested 24, 48, and 72 h after transfection.

Constitutive PD-L1 expression assay
The constitutive PD-L1 expression of different BC cell lines under standardized culture conditions was analyzed by flow cytometry.MCF-7, MDA-MB-231, and BT-549 cell lines (1 × 10 5 cells/well) were seeded into a 12-well plate, 24 h before transfection.BC cell line and MCF-10 cell line, as negative control, were transfected with miR-561-3p.After 48 h, the expression of cell-surface PD-L1 was investigated.In the first step, the cells were incubated with anti-CD16/32 (eBioscience, for mouse cells) to block the fc receptors.Then, the cells were stained with a LIVE/DEAD viability dye (Thermofisher Scientific) for 10 min at 4 °C in FACS buffer (PBS, 0.5% BSA, 2mM EDTA).In the next step, the cells were incubated with the PE anti-human CD274 (B7-H1, PD-L1) antibody (Biolegend, 329705) for 30 min at 4 °C and then analyzed with FlowJo 10.6.2 / 10.0.7 R2 software.www.nature.com/scientificreports/

Apoptosis
MCF-7, MDA-MB-231, and BT-549 cell lines (1 × 10 5 cells/well) were seeded into a 12-well plate, 24h before transfection.BC cell lines were transfected with scrambled oligonucleotides and miR-561-3p using HiPerFect transfection reagent (QIAGEN.Germany), according to the manufacturer's protocol.After 48 h, the cellular apoptotic rate was evaluated via Annexin V-FITC/PI staining kit (Biolegend.Austria) based on the provided instructions The results were analyzed using flow cytometry (FlowJo 10.6.2 / 10.0.7 R2 software).Each sample was run in triplicate.

Statistical analysis data
RT-qPCR results were analyzed using REST ® , 2009 Software.All data were presented as mean ± SE.P values less than 0.05 were considered to be significant.Statistical analysis was performed using GraphPad Prism V.8 software.The t-test was used to compare cancerous and non-cancerous breast cells and samples.Differences between groups were analyzed by analysis of variance (ANOVA) when more than two groups were compared.Correlation analysis was performed using the Pearson test . https://doi.org/10.1038/s41598-024-56511-6

Figure 2 .
Figure 2. miR-561-3p directly targets ZEB1, MYC, and HIF1A genes.The 3′-UTR s sequence of the (A) ZEB1, (B) MYC, and (C) HIF1A 'genes and the miR-561-3p binding sites.Prediction of the target genes for miR-561-3p using an online miR target prediction database.(D) MDA-MB-231, (E) BT-549, (F) MCF-7 cells were transfected with the recombinant psiCHECK TM -2 vector harboring the ZEB1, MYC, and HIF1A 3′-UTRs, and vectors cloned by miR-561-3p or scrambled microRNA.Dual-Luciferase assays were performed for each cell line and decrease in relative Renilla luciferase activity was observed, but relative Renilla luciferase activity was not significantly changed in the cells treated with the scrambled microRNA.***P < 0.001, ns non-significant, n = 3.Data represent as mean ± SE from three independent performed experiments.
adjacent normal tissues were obtained from khatamol Anbia Hospital and instantly frozen in liquid nitrogen, and stored in −70 °C.Clinical data of the patients, including sex, age, tumor differentiation, lymph node metastasis, and clinical data were obtained from the hospital records (Table1).

Table 1 .
Demographic and clinical characteristics of the breast cancer patients.

Table 2 .
30imer sequence for real-time PCR analysis.Statistical analysis for relative mRNA expression was performed by the Relative Expression Software Tool (REST) proposed by Pfaffl30.Fold change and P-value (< 0.05) were determined by REST, which considered all software requirements.All PCR assays displayed efficiencies between 1.8 and 2.0.Primers for real-time PCR were designed by Oligo version 7.56 and check them for specificity by using Primer-BLAST software (http:// blast.ncbi.nlm.nih.gov/9((Table2).All oligos were purchased from Cinaclon Company(Iran,Tehran).
Table3indicate the XhoI and NotI restriction sites within the designed primers.Pfu DNA polymerase (Fermentas, EP0501) was used for accurate amplification of the 3′-UTRs according to the manufacturer's protocol.The PCR products and special scrambled sequence (AAG CTT CAT AAG GCG CAT AGC), as a negative control, were cloned into psiCHECK™-2 vector (Promega, C8021) immediately downstream of the stop codon of the renilla gene.Insertion of the PCR products into psiCHECK™-2 vector was confirmed by colony PCR, double digestion with NotI (Fermentas, ER0571) and Xho1 (Fermentas, ER0691) restriction enzymes, and sequencing (Niagen Noor Medical Genetics Laboratory, Tehran, Iran).Luciferase activity was measured with the Dual-Glo ® Luciferase Assay System (Promega, E2940) 48 h after transfection.The multiwell plate luminometer Renilla luciferase activity was normalized to that of firefly luciferase.

Table 3 .
Primer sequence for 3-UTRs PCR analysis (the XhoI and NotI restriction sites were highlighted in bold).